Primary malignant central nervous system (CNS) neoplasms, particularly glioblastomas, are highly fatal due to their aggressive and widespread infiltration of the brain and resistance to anti-cancer treatments. Although progress has been made in unraveling the pathological mechanisms underlying CNS cancers as well as other cancer types, tumor specific therapeutic approaches and methods of diagnosis have been largely elusive.
The invention features a method for diagnosing a malignant neoplasm in a mammal by contacting a bodily fluid from the mammal with an antibody which binds to an human aspartyl (asparaginyl) beta-hydroxylase (HAAH) polypeptide under conditions sufficient to form an antigen-antibody complex and detecting the antigen-antibody complex (for the purposes of this specification, HAAH polypeptide refers to the amino acid sequence of SEQ ID NO:2 and HAAH cDNA refers to the nucleotide sequence of SEQ ID NO:3). Malignant neoplasms detected in this manner include those derived from endodermal tissue, e.g., colon cancer, breast cancer, pancreatic cancer, liver cancer, and cancer of the bile ducts. Neoplasms of the central nervous system (CNS) such as primary malignant CNS neoplasms of both neuronal and glial cell origin and metastatic CNS neoplasms are also detected. Patient derived tissue samples, e.g., biopsies of solid tumors, as well as bodily fluids such as a CNS-derived bodily fluid, blood, serum, urine, saliva, sputum, lung effusion, and ascites fluid, are contacted with an HAAH-specific antibody.
The invention includes a method of eliciting an immune response or confering an immune response to a tumor cell, e.g., a brain tumor, in a mammal by administering to a mammal an antibody which binds to HAAH or a polynucleotide encoding such an antibody. Preferably, the antibody binds to a site in an extracellular domain (e.g., a site within residues 1-700) of HAAH. The antibody binds to an ectodomain of HAAH (residues 19-75 of SEQ ID NO:2). More preferably, the antibody binds to a catalytic domain of HAAH, e.g., amino acids 650-700 of SEQ ID NO:2. For example, FB50 binds to a polypeptide with the amino acid sequence NPVEDS (residues 286-291 of SEQ ID NO:2). Monoclonal antibody HBOH1 binds to a polypeptide with the amino acid sequenc QPWWTPK (residues 573-579 of SEQ ID NO:2), and monoclonal antibody HBOH-2 binds to a polypeptide containing the amino acid sequence LPEDENLR (residues 613-620 of SEQ ID NO:2). The foregoing antigenic epitopes of HAAH are located on the cell surface of malignant cells. Other HAAH-specific antibodies suitable for passive immunization include 5C7, 5E9, 19B, 48A, 74A, 78A, 86A, HA238A, HA221, HA 239, HA241, HA329, and HA355.
The antibody to be administered is a heterodimeric antibody, a single chain antibody, or a high affinity single chain antibody. By high affinity is meant that the antigen-specific binding affinity of the antibody has a Kd in the nanomolar range. Preferably, the binding affinity is in the range of 100 pM or higher affinity. For example, the antibody, antibody fragment, or single chain antibody has an antigen-specific binding affinity in the range of 10xe2x88x9210 to 10xe2x88x9215 molar.
The antibody, or fragment thereof, activates complement in a patient treated with the antibody. Preferably, the antibody mediates antibody-dependent cytotoxicity of tumor cells in the patient treated with the antibody. The antibody, or fragment thereof, is administered alone or conjugated to a cytotoxic agent. In the latter case, binding of the antibody to a tumor cell results in impairment or death of the cell, thereby reducing tumor load. The antibody is conjugated to a radiochemical, or a chemical tag which sensitizes the cell to which it is bound to radiation or laser-mediated killing.
Also within the invention, are methods of inducing an HAAH-specific immune response to reduce tumor growth by active immunization. The method involves administering to a mammal an HAAH polypeptide, e.g., a polypeptide containing the amino acid sequence of SEQ ID NO:2. Immunogenic HAAH fragments are also administered to generate an immune response to a particular portion of HAAH. For example, to generate an antibody response to HAAH on the surface of cells, a polypeptide containing an extracellular domain of HAAH (but lacking an intracellular domain of HAAH) is administered. To generate antibodies, which inhibit HAAH activity, the individual is immunized with a polypeptide containing a catalytic domain of HAAH (e.g., amino acids 650-700 of SEQ ID NO:2). Optionally, the polypeptide compositions contain a clinically-acceptable adjuvant compound. Such adjuvants are generally known in the art, and include oil-emulsions, Freunds Complete and Incomplete adjuvant, Vitamin E, aluminum salts or gels, such as aluminum hydroxide, -oxide or -phosphate, saponins, polymers based on polyacrylic acid, such as carbopols, non-ionic block polymers, fatty acid amines, such as avridin and DDA, polymers based on dextran, such as dextran sulphate and DEAE dextran, muramyldipeptides, ISCOMs (immune stimulating complexes, e.g., as described in European Patent EP 109942), biodegradable microcapsules, liposomes, bacterial immune stimulators, such as MDP and LPS, and glucans. Other adjuvant compounds are known in the art, e.g., described in Altman and Dixon, 1989, Advances in Veterinary Science and Comparative Medicine 33: 301-343). Alum is preferred for human use.
An HAAH-specific immune response is also induced by administering to a mammal a polynucleotide composition encoding an HAAH polypeptide, or a degenerate variant of the HAAH-encoding polynucleotide. For example, the polynucleotide contains the nucleotide sequence of SEQ ID NO:3, or a degenerate variant thereof, or a fragment thereof encoding a specific immunogenic domain of HAAH. Preferably, the HAAH polypeptide encoded by the polynucleotide (or directly administered polypeptide) is enzymatically nonfunctional. More preferably, the HAAH polynucleotide encodes an HAAH polypeptide that is secreted, e.g., the construct contains a signal sequence for transport out of the cell and into an extracellular space. The HAAH polypeptide lacks an essential histidine. The HAAH polypeptide is a truncated HAAH, which contains the first 650 amino acids of SEQ ID NO:2.
Optionally, the polynucleotide composition contains a transfection-enhancing agent, such as a precipitating agent or a lipid. Preferably, the encoded HAAH polyeptide contains the amino acid sequence of SEQ ID NO:2 (full-length HAAH) or a fragment thereof, which contains an extracellular domain of HAAH and lacks an intracellular domain of HAAH. Preferably, the polynucleotide contains a catalytic domain of HAAH. The HAAH-encoding sequences are operably-linked to a promoter and other regulatory sequences for expression of the polypeptides in target cells. The polypeptide may be directed intracellularly or marked for extracellular expression, or secretion. The polynucleotide directs expression in a target cell, which expresses appropriate accessory molecules for antigen presentation, e.g., major histocompatibility antigens.
Methods for diagnosis include detecting a tumor cell in bodily fluids as well as detecting a tumor cell in tissue (in vivo or ex vivo). For example, a biopsied tissue is contacted with an HAAH-specific antibody and antibody binding measured. Whole body diagnostic imaging may be carried out to detect microtumors undetectable using conventional diagnostic methods. Accordingly, a method for diagnosing a neoplasm in a mammal is carried out by contacting a tissue, e.g., a lymph node, of a mammal with a detectably-labeled antibody which binds to HAAH. An increase in the level of antibody binding at a tissue site compared to the level of binding to a normal nonneoplastic tissue indicates the presence of a neoplasm at the tissue site. For detection purposes, the antibody (or HAAH-binding fragment thereof) is labeled with a non-radioactive tag, a radioactive compound, or a colorimetric agent. For example, the antibody or antibody fragment is tagged with 125I, 99Tc, Gd+++, or Fe++. Green fluorescent protein is used as a calorimetric tag.
The invention also includes a soluble fragment of HAAH. The soluble HAAH polypeptide contains an extracellular domain and optionally lacks part or all of the cytoplasmic domain or transmembrane domain of HAAH. In one example, the fragment lacks residues 660-758 of SEQ ID NO:2. In another example, the fragment lacks residues 679-697 (His motif) of SEQ ID NO:2. In yet another example, the fragment, lacks at least one residue of SEQ ID NO:2, the residue being selected from the group consisting of residue 661, 662, 663, 670, 671, 672, and 673. An HAAH fragment is an HAAH polypeptide, the length of which is less than that of a fill-length HAAH protein. The full-length HAAH protein is shown in Table 1.
Diagnostic kits are also encompassed by the invention. For example, a kit for detecting a tumor cell contains an antibody, or fragment thereof, which binds to HAAH. The kit optionally contains a means for detecting binding of the antibody to the tumor cell. For example, the kit contains a detectable marker, e.g., a nonradioactive marker such as Gd+++ or Fe++ or a radioactive compound. The kit may also contain instructions for use, a standard reagent for determining positive antibody binding, or a negative control for determining lack of antibody binding. The components are packaged together in a kit.
The assay format described herein is useful to generate temporal data used for prognosis of malignant disease. A method for prognosis of a malignant neoplasm of a mammal is carried out by (a) contacting a bodily fluid from the mammal with an antibody which binds to an HAAH polypeptide under conditions sufficient to form an antigen-antibody complex and detecting the antigen-antibody complex; (b) quantitating the amount of complex to determine the level of HAAH in the fluid; and (c) comparing the level of HAAH in the fluid with a normal control level of HAAH. An increasing level of HAAH over time indicates a progressive worsening of the disease, and therefore, an adverse prognosis.
The invention also includes an antibody which binds to HAAH. The antibody preferably binds to a site in the carboxyterminal catalytic domain of HAAH. Alternatively, the antibody binds to an epitope that is exposed on the surface of the cell. The antibody is a polyclonal antisera or monoclonal antibody. The invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin. Preferably the antibody is a monoclonal antibody such as FB50, 5C7, 5E9, 19B, 48A, 74A, 78A, 86A, HA238A, HA221, HA 239, HA241, HA329, or HA355. Antibodies which bind to the same epitopes as those monoclonal antibodies are also within the invention.
An HAAH-specific intrabody is a recombinant single chain HAAH-specific antibody that is expressed inside a target cell, e.g., tumor cell. Such an intrabody binds to endogenous intracellular HAAH and inhibits HAAH enzymatic activity or prevents HAAH from binding to an intracellular ligand. HAAH-specific intrabodies inhibit intracellular signal transduction, and as a result, inhibit growth of tumors which overexpress HAAH.
A kit for diagnosis of a tumor in a mammal contains an HAAH-specific antibody. The diagnostic assay kit is preferentially formulated in a standard two-antibody binding format in which one HAAH-specific antibody captures HAAH in a patient sample and another HAAH-specific antibody is used to detect captured HAAH. For example, the capture antibody is immobilized on a solid phase, e.g., an assay plate, an assay well, a nitrocellulose membrane, a bead, a dipstick, or a component of an elution column. The second antibody, i.e., the detection antibody, is typically tagged with a detectable label such as a calorimetric agent or radioisotope.
Also within the invention is a method of inhibiting tumor growth in a mammal, which is carried out by administering to the mammal a compound which inhibits expression or enzymatic activity of HAAH. Preferably, the compound is substantially pure nucleic acid molecule such as an HAAH antisense DNA, the sequence of which is complementary to a coding sequence of HAAH. Expression of HAAH is inhibited by contacting mammalian cells, e.g., tumor cells, with HAAH antisense DNA or RNA, e.g., a synthetic HAAH antisense oligonucleotide. The sequence of the antisense is complementary to a coding or noncoding region of a HAAH gene. For example, the sequence is complementary to a nucleotide sequence in the 5xe2x80x2 untranslated region of a HAAH gene. Examples of HAAH antisense oligonucleotides which inhibit HAAH expression in mammalian cells include oligonucleotides containing SEQ ID NO:10, 11, or 12. An HAAH antisense nucleic acid is introduced into glioblastoma cells or other tumor cells which overexpress HAAH. Binding of the antisense nucleic acid to an HAAH transcript in the target cell results in a reduction in HAAH production by the cell. By the term xe2x80x9cantisense nucleic acidxe2x80x9d is meant a nucleic acid (RNA or DNA) which is complementary to a portion of an mRNA, and which hybridizes to and prevents translation of the mRNA. Preferably, the antisense DNA is complementary to the 5xe2x80x2 regulatory sequence or the 5xe2x80x2 portion of the coding sequence of HAAH mRNA (e.g., a sequence encoding a signal peptide or a sequence within exon 1 of the HAAH gene). Standard techniques of introducing antisense DNA into the cell may be used, including those in which antisense DNA is a template from which an antisense RNA is transcribed. The method is to treat tumors in which expression of HAAH is upregulated, e.g., as a result of malignant transformation of the cells. The length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring HAAH transcript. Preferably, the length is between 10 and 50 nucleotides, inclusive. More preferably, the length is between 10 and 20 nucleotides, inclusive.
By xe2x80x9csubstantially pure DNA or RNAxe2x80x9d is meant that the nucleic acid is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank a HAAH gene. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a procaryote or eucaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequence such as a nucleic acid encoding an chimeric polypeptide, e.g., one encoding an antibody fragment linked to a cytotoxic polypeptide. Alternatively, HAAH expression is inhibited by administering a ribozyme or a compound which inhibits binding of Fos or Jun to an HAAH promoter sequence.
Compounds, which inhibit an enzymatic activity of HAAH, are useful to inhibit tumor growth in a mammal. By enzymatic activity of HAAH is meant hydroxylation of an epidermal growth factor (EGF)-like domain of a polypeptide. For example an EGF-like domain has the consensus sequence CX7CX4CX10CXCX8C (SEQ ID NO:1). HAAH hydroxylase activity is inhibited intracellularly. For example, a dominant negative mutant of HAAH (or a nucleic acid encoding such a mutant) is administered. The dominant negative HAAH mutant contains a mutation which changes a ferrous iron binding site from histidine of a naturally-occurring HAAH sequence to a non-iron-binding amino acid, thereby abolishing the hydroxylase activity of HAAH. The histidine to be mutated, e.g., deleted or substituted, is located in the carboxyterminal catalytic domain of HAAH. For example, the mutation is located between amino acids 650-700 (such as the His motif, underlined sequence of SEQ ID NO:2) the native HAAH sequence. For example, the mutation is at residues 671, 675, 679, or 690 of SEQ ID NO:2. An HAAH-specific intrabody is also useful to bind to HAAH and inhibit intracellular HAAH enzymatic activity, e.g., by binding to an epitope in the catalytic domain of HAAH. Other compounds such as L-mimosine or hydroxypyridone are administered directly into a tumor site or systemically to inhibit HAAH hydroxylase activity.
For example, a compound which inhibits HAAH hydroxylation is a polypeptide that binds a HAAH ligand but does not transduce an intracellular signal or an polypeptide which contains a mutation in the catalytic site of HAAH. Such a polypeptide contains an amino acid sequence that is at least 50% identical to a naturally-occurring HAAH amino acid sequence or a fragment thereof and which has the ability to inhibit HAAH hydroxylation of substrates containing an EGF-like repeat sequence. More preferably, the polypeptide contains an amino acid sequence that is at least 75%, more preferably at least 85%, more preferably at least 95% identical to SEQ ID NO:2.
A substantially pure HAAH polypeptide or HAAH-derived polypeptide such as a mutated HAAH polypeptide is preferably obtained by expression of a recombinant nucleic acid encoding the polypeptide or by chemically synthesizing the protein. A polypeptide or protein is substantially pure when it is separated from those contaminants which accompany it in its natural state (proteins and other naturally-occurring organic molecules). Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, HAAH. Purity is measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eucaryote but produced in E. coli or another procaryote, or in a eucaryote other than that from which the polypeptide was originally derived.
Nucleic acid molecules which encode such HAAH or HAAH-derived polypeptides are also within the invention.
Methods of inhibiting tumor growth also include administering a compound which inhibits HAAH hydroxylation of a NOTCH polypeptide. For example, the compound inhibits hydroxylation of an EGF-like cysteine-rich repeat sequence in a NOTCH polypeptide, e.g., one containing the consensus sequence CDXXXCXXKXGNGXCDXXCNNAACXXDGXDC (SEQ ID NO:4). Polypeptides containing an EGF-like cysteine-rich repeat sequence are administered to block hydroxylation of endogenous NOTCH.
Growth of a tumor which overexpresses HAAH is also inhibited by administering a compound which inhibits signal transduction through the insulin receptor substrate (IRS) signal transduction pathway. Preferably the compound inhibits IRS phosphorylation. For example, the compound is a peptide or non-peptide compound which binds to and inhibits phosphorylation at residues 46, 465, 551, 612, 632, 662, 732, 941, 989, or 1012 of SEQ ID NO:5. Compounds include polypeptides such those which block an IRS phosphorylation site such as a Glu/Tyr site. Antibodies such as those which bind to a carboxyterminal domain of IRS containing a phosphorylation site block IRS phosphorylation, and as a consequence, signal transduction along the pathway. Inhibition of IRS phosphorylation in turn leads to inhibition of cell proliferation. Other compounds which inhibit IRS phosphorylation include vitamin D analogue EB1089 and Wortmannin.
HAAH-overproducing tumor cells were shown to express HAAH both intracellularly and on the surface of the tumor cell. Accordingly, a method of killing a tumor cell is carried out by contacting such a tumor cell with a cytotoxic agent linked to an HAAH-specific antibody. The HAAH-specific antibody (antibody fragment, or ligand which binds to extracellular HAAH) directs the chimeric polypeptide to the surface of the tumor cell allowing the cytotoxic agent to damage or kill the tumor cell to which the antibody is bound. The monoclonal antibody binds to an epitope of HAAH such as an epitope exposed on the surface of the cell or in the catalytic site of HAAH. The cytotoxic composition preferentially kills tumor cells compared to non-tumor cell.
Screening methods to identify anti-tumor agents which inhibit the growth of tumors which overexpress HAAH are also within the invention. A screening method used to determine whether a candidate compound inhibits HAAH enzymatic activity includes the following steps: (a) providing a HAAH polypeptide, e.g., a polypeptide which contains the carboxyterminal catalytic site of HAAH; (b) providing a polypeptide comprising an EGF-like domain; (c) contacting the HAAH polypeptide or the EGF-like polypeptide with the candidate compound; and (d) determining hydroxylation of the EGF-like polypeptide of step (b). A decrease in hydroxylation in the presence of the candidate compound compared to that in the absence of the compound indicates that the compound inhibits HAAH hydroxylation of EGF-like domains in proteins such as NOTCH.
Anti-tumor agents which inhibit HAAH activation of NOTCH are identified by (a) providing a cell expressing HAAH; (b) contacting the cell with a candidate compound; and (c) measuring translocation of activated NOTCH to the nucleus of the cell. Translocation is measured by using a reagent such as an antibody which binds to a 110 kDa activation fragment of NOTCH. A decrease in translocation in the presence of the candidate compound compared to that in the absence of the compound indicates that the compound inhibits HAAH activation of NOTCH, thereby inhibiting NOTCH-mediated signal transduction and proliferation of HAAH-overexpressing tumor cells.
Nucleotide and amino acid comparisons described herein were carried out using the Lasergene software package (DNASTAR, Inc., Madison, Wis.). The MegAlign module used was the Clustal V method (Higgins et al., 1989, CABIOS 5(2):151-153). The parameter used were gap penalty 10, gap length penalty 10.
Hybridization is carried out using standard techniques, such as those described in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, 1989). xe2x80x9cHigh stringencyxe2x80x9d refers to nucleic acid hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65xc2x0 C. at a salt concentration of 0.1xc3x97SSC. xe2x80x9cLowxe2x80x9d to xe2x80x9cmoderatexe2x80x9d stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g., wash conditions of less than 60xc2x0 C. at a salt concentration of 1.0xc3x97SSC. For example, high stringency conditions include hybridization at 42xc2x0 C. in the presence of 50% formamide; a first wash at 65xc2x0 C. in the presence of 2xc3x97SSC and 1% SDS; followed by a second wash at 65xc2x0 C. in the presence of 0.1%xc3x97SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an HAAH gene sequence are detected by, for example, hybridization at about 42xc2x0 C. in the absence of formamide; a first wash at 42xc2x0 C., 6xc3x97SSC, and 1% SDS; and a second wash at 50xc2x0 C., 6xc3x97SSC, and 1% SDS.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.